The disclosures referred to herein to illustrate the background of the invention and to provide additional detail with respect to its practice are incorporated herein by reference. For convenience, the disclosures are referenced in the following text and respectively grouped in the appended bibliography.
Retroviral vectors are the most efficient tools to introduce genes into vertebrate cells. Clinical experiments have been conducted to use retrovirus vectors to cure a genetic disease in humans (adenosine deaminase (ADA) deficiency). Besides correcting inborn errors of metabolism, gene therapy is also being tested in clinical trials to cure cancer and various other diseases (Science 1992, Vol. 258, pp. 744-746).
Retroviral vectors are basically retroviral particles that contain a genome in which all viral protein coding sequences have been replaced with the gene(s) of interest. As a result, such viruses cannot further replicate after one round of infection. Retroviral vector particles are produced by helper cells (FIG. 1). Such helper cells are cell lines that contain plasmid constructs which express all retroviral proteins necessary for replication. After transfection of the vector genome into such helper cells, the vector genome is encapsidated into virus particles (due the presence of specific encapsidation sequences). Virus particles are released from the helper cell carrying a genome containing only the gene(s) of interest (FIG. 1). In the last decade, several retroviral vector systems, derived from chicken or murine retroviruses, have been developed for the expression of various genes (for reviews see Temin, 1987; Gilboa, 1990).
Retroviral vectors have several limitations. Besides the limited genome size that can be encapsidated into viral particles, the most limiting factor for the application of retroviral vectors is the restricted host range of the vector particle. Some retroviruses can only infect cells of one species (ecotropic retroviruses) or even only one cell-type of one species (e.g., HIV). Other retroviruses have a very broad host range and can infect many different types of tissues of many different species (amphotropic retroviruses).
The initial step of retroviral infection is the binding of the viral envelope (env) glycoprotein to specific cell membrane receptors, the nature of which is unknown for most retroviruses. However, the interaction of the viral env protein with the cell surface receptor is very specific and determines cell-type specificity of a particular virus (Weiss et al, 1985). The envelope protein of all known retroviruses is made up of two associated peptides, (e.g., gp70 and p20(E) in SNV). These peptides are derived by proteolytic cleavage from the same precursor (gPR90env) encoded by the retroviral env gene. One peptide p20(E), also termed TM, anchors the protein in the membrane of the virus and, as shown with HIV, mediates the fusion of the virus and cell membranes. The second peptide gp70, also termed SU, mediates the binding of the virus to its receptor and, therefore, determines the host range (Weiss et al., 1985; Varmus and Brown, 1989).
Data obtained with several retroviruses indicate that the retroviral envelope protein forms trimers or tetramers. The formation of trimers appears to be mediated by the TM peptide (reviewed in Hunter, E. et al., 1990). Targeting envelopes retain TM in order to (i) maintain a membrane fusion function and (ii) maintain oligomerization. However, since X-ray pictures are not available, it is unclear whether or to what degree the construction of targeting-molecules impaired the structure of the membrane fusion domain.